Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species

Abstract

Introduction: Administration of mesenchymal stem cells (MSCs) represents a promising treatment option for patients suffering from immunological and degenerative disorders. Accumulating evidence indicates that the healing effects of MSCs are mainly related to unique paracrine properties, opening opportunities for secretome-based therapies. Apart from soluble factors, MSCs release functional small RNAs via extracellular vesicles (EVs) that seem to convey essential features of MSCs. Here we set out to characterize the full small RNAome of MSC-produced exosomes.

Methods: We set up a protocol for isolating exosomes released by early passage adipose- (ASC) and bone marrow-MSCs (BMSC) and characterized them via electron microscopy, protein analysis and small RNA-sequencing. We developed a bioinformatics pipeline to define the exosome-enclosed RNA species and performed the first complete small RNA characterization of BMSCs and ASCs and their corresponding exosomes in biological replicates.

Results: Our analysis revealed that primary ASCs and BMSCs have highly similar small RNA expression profiles dominated by miRNAs and snoRNAs (together 64-71 %), of which 150-200 miRNAs are present at physiological levels. In contrast, the miRNA pool in MSC exosomes is only 2-5 % of the total small RNAome and is dominated by a minor subset of miRNAs. Nevertheless, the miRNAs in exosomes do not merely reflect the cellular content and a defined set of miRNAs are overrepresented in exosomes compared to the cell of origin. Moreover, multiple highly expressed miRNAs are precluded from exosomal sorting, consistent with the notion that these miRNAs are involved in functional repression of RNA targets. While ASC and BMSC exosomes are similar in RNA class distribution and composition, we observed striking differences in the sorting of evolutionary conserved tRNA species that seems associated with the differentiation status of MSCs, as defined by Sox2, POU5F1A/B and Nanog expression.

Conclusions: We demonstrate that primary MSCs release small RNAs via exosomes, which are increasingly implicated in intercellular communications. tRNAs species, and in particular tRNA halves, are preferentially released and their specific sorting into exosomes is related to MSC tissue origin and stemness. These findings may help to understand how MSCs impact neighboring or distant cells with possible consequences for their therapeutic usage.

Fig. 1

ASCs and BMSCs contain relatively few MVBs and release exosome-like vesicles enriched in small RNAs. a Immunofluorescent staining of CD63 (top left) and EEA1 (bottom left), and ultrastructure of MVB-like endosomes (right) in ASCs and BMSCs. b Transmission electron microscopy micrographs of exosomes isolated from ASCs and BMSCs (left); and western blot for CD63, CD81, and cytochrome C in cells and corresponding exosomes (right). c Bioanalyzer small RNA profile of cells and exosomes showing enrichment of 20–70 nucleotide small RNAs in exosomes. ASC adipose-derived mesenchymal stem cell, BMSC bone marrow-derived mesenchymal stem cell, EEA1 early endosome antigen A1, exo exosome

Fig. 2

MSCs and their exosomes display a different small RNA composition. a Length distribution of RNAseq aligned reads in ASCs and BMSCs and corresponding exosomes (one representative donor). b Unsupervised hierarchical clustering analysis of MSCs and exosomes based on the total small RNA content. ASC adipose-derived mesenchymal stem cell, BMSC bone marrow-derived mesenchymal stem cell, exo exosome

Fig. 3

Exosomes released by MSCs at different stages of differentiation correlate moderately with each other. a Correlation matrix of MSC and exosome samples. b Relative expression levels of early osteogenic differentiation (top) and multipotency (bottom) genes analyzed by quantitative PCR. Data were normalized to GAPDH. BMSC II express higher levels of Sox2 (ANOVA test: p = 0.006; Fisher’s PLSD test: p = 0.02 BMSC II vs. ASC, p = 0.03 BMSC II vs. BMSC I), POU5F1A/B (ANOVA test: p = 0.01; Fisher’s PLSD test: p = 0.03 BMSC II vs. ASC, p = 0.01 BMSC II vs. BMSC I) and Nanog (ANOVA test: p = 0.03; Fisher’s PLSD test: p = 0.056 BMSC II vs. ASC) compared with the other subtypes. ALP alkaline phosphatase, ANOVA analysis of variance, ASC adipose-derived mesenchymal stem cell, BMSC bone marrow-derived mesenchymal stem cell, COL1A1 collagen type 1 alpha 1, exo exosome, GAPDH glyceraldehyde 3-phosphate dehydrogenase, Fisher's PLSD Fisher's Protected Least Significant Difference

Fig. 4

Mesenchymal stem cells and their exosomes have a different RNA class distribution. a Relative distribution of overrepresented RNA species in MSCs and exosomes. b Differential representation of RNA classes in MSC exosomes versus cells ordered by logFC. ASC adipose-derived mesenchymal stem cell, BMSC bone marrow-derived mesenchymal stem cell, exo exosome, snoRNA small nucleolar RNA, logFC log fold-change, FDR false discovery rate

Fig. 5

MSCs and their exosomes display a different miRNA repertoire. a Unsupervised hierarchical clustering analysis of MSCs and their exosomes based on the miRNA content. b Length distribution of miRNA reads in ASC and BMSC I and BMSC II cells and exosomes (one representative donor). c Sequence coverage of highly represented miRNA genes (based on UCSC genome browser custom tracks) showing a predominant presence of mature forms (one or both miRNA arms) in MSCs and exosomes. Y axis indicates the normalized counts (rpm). ASC adipose-derived mesenchymal stem cell, BMSC bone marrow-derived mesenchymal stem cell, exo exosome

Fig. 6

MSC exosomes selectively incorporate specific miRNAs. a Relative proportion of miRNAs in the repertoire of total miRNA reads. The five most abundant miRNAs represent 50 % of the total miRNA reads. b Overrepresented and c underrepresented miRNAs in MSC exosomes as compared with producing cells (LogFC >3; FDR <0.0002). ASC adipose-derived mesenchymal stem cell, BMSC bone marrow-derived mesenchymal stem cell, exo exosome

Fig. 7

tRNA-derived RNA fragments are highly represented in MSC exosomes. a Relative distribution of highly represented tRNAs in MSCs and respective exosomes. b Length distribution of tRNA sequencing reads in ASC and BMSC I and II cells and exosomes. c Sequence coverage of highly represented tRNA genes (based on UCSC genome browser custom tracks). Y axis indicates the normalized counts (rpm). ASC adipose-derived mesenchymal stem cell, BMSC bone marrow-derived mesenchymal stem cell, exo exosome